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P W Madden

Publications and source records attributed to P W Madden.

5 recordsLinked to original sources
PubMed1989

The extent of immunological privilege of orthotopic corneal grafts in the inbred rat.

Corneal grafts are believed to enjoy a degree of "immunological privilege" primarily due to the avascularity of the recipient bed. In this study orthotopic full-thickness corneal grafts were carried out in the inbred rat, using a technique that is a close model of corneal grafting in humans. The survival times of corneal grafts on nonvascularized beds of 28 fully allogeneic strain combinations were determined without the use of immunosuppression. Some combinations were rejected rapidly, e.g. DA (RT1a) into BN (RT1n) with a mean survival time (MST) +/- SD of 7.8 +/- 1.3 days, and some at a moderate rate, e.g. AO (RT1u) into LEW (RT1l) with an MST of 23.1 +/- 10.0 days, whereas in other cases survival was indefinite, e.g. WAG (RT1u) into PVG (RT1c), an MST of greater than 100 days. Orthotopic corneal grafts on nonvascularized beds between DA and AO parents and the F1, followed the basic rules of transplantation genetics. In addition, the rate of graft rejection was significantly faster (P less than 0.001) with corneal grafts from DA into AO placed onto a vascularized compared with a nonvascularized corneal bed (MST of 6.8 +/- 2.4 or 12.1 +/- 4.0 days respectively). The rate of rejection of corneas on a vascularized bed was at a similar rate to that of orthotopic skin or heterotopic auxiliary heart grafts. The results indicate that the fate of a corneal allograft on a nonvascularized bed is dependent upon the particular combination of donor and recipient strain. No consistent association was observed between any donor or recipient RT1 haplotype and survival; this suggests that non-RT1 background genes may play a role in the survival of corneal grafts.

Animals↗
PubMed1989

Hypothermic preservation of corneas in a hyperkalaemic solution (CPTES): II. Extended storage in the presence of chondroitin sulphate.

Periods of preservation for donor corneas, even for short times, are necessary to facilitate optimum conditions in penetrating keratoplasty. However, current techniques for corneal storage at low temperatures may not provide optimal conditions for maintaining tissue integrity. In particular, the ionic composition of the storage medium has received little attention since it has been assumed throughout that the normal complement of ions in tissue culture media will also be suitable for preservation at reduced temperatures. This study extends our previous investigations on the merits of using CPTES (corneal-potassium-TES), a potassium-rich balanced salt solution containing an impermeant anionic pH buffer (TES), as a storage solution specifically designed to prevent the loss of intracellular potassium and minimise endothelial cell swelling during the time that the normal regulatory processes are switched off. The effect of adding the natural polymer chondroitin sulphate (CS) as a colloid osmotic agent to the hyperkalaemic storage medium is now examined. Corneas stored in CPTES containing 2.5% chondroitin sulphate retained a very high level of structural and functional integrity after three, five, and seven days storage at 0 degrees C; furthermore, stromal swelling was restricted to only 21%. All corneas stored in CPTES + 2.5% CS showed active endothelial function by thinning efficiently at rates that were greater than those previously reported for rabbit corneas stored for similar lengths of time in either M-K medium or K-sol. The zwitterionic buffers TES and HEPES were interchangeable in the hyperkalaemic solution and were non-toxic to corneal endothelium at a concentration of 100 mM. These compounds offer excellent pH buffering in bicarbonate-free medium.

Animals↗
PubMed1989

The assessment of endothelial integrity by scanning electron microscopy and fluorescein diacetate staining following treatment with cryoprotective additives.

As part of the development of methods of corneal cryostorage for transplantation, a toxicity study was carried out on the rabbit corneal endothelium using four cryoprotective additives (CPA's) 1) dimethyl sulphoxide (Me2SO), 2) propane-1,2-diol (PG), 3) glycerol (GLY), 4) polyvinylpyrrolidone (PVP). A fifth group, based upon a CPA combination of Me2SO and PVP, was used to characterize both the assays, and the response of the endothelial layer to osmotic stress. The effect upon the cell membrane was assessed using scanning electron microscopy (SEM) and fluorescein diacetate with ethidium bromide staining (FDA/EB). Two sampling points were used, one immediately after treatment and the other following an incubation period. Calculations were performed to predict the maximum relative volume of cells during CPA exchange. Immediately following serial addition and removal of 2 or 3 mol/L (M) PG or GLY, the cells exhibited adverse morphological changes shown with SEM, and the proportion of intact cells judged by FDA/EB staining was significantly reduced when CPA equilibration was performed at 37 degrees C rather than at 20 degrees C. A 3M Me2SO concentration gave less morphological change than 3M PG or GLY, but even after treatment with 4M Me2SO more than 95% cells were judged intact by FDA/EB staining. PVP at 40% w/v showed minimal damage with both assays, and the fifth experimental group suggested that PVP may protect from injury during hypotonic stress. With all groups, the integrity of the cell layer recovered during incubation, so that for each sample the percentage of intact cells was high. However, although confluency was often restored following incubation, total cell density was usually reduced. The results indicate that serial addition and removal of 3M Me2SO is tolerated by the cornea, whereas PG or GLY cannot be used at 2 or 3M without inducing osmotic damage. There was low toxicity to PVP, and it was an effective osmotic buffer.

Animals↗
PubMed1987

The evaluation of endothelial damage following corneal storage: a comparison of staining methods and the value of scanning electron microscopy.

Staining techniques and scanning electron microscopy (SEM) were used to assess damage to the endothelial cells following corneal storage. The aim was to establish whether these assays are useful in the assessment of endothelial integrity following corneal storage. Four groups ensured a range of normal and damaged endothelial cells: 1) fresh; 2) stored for 7 days in a moist chamber; 3) stored by the cryopreservation method of Capella and Kaufman; 4) damaged by rapid freezing with a cryoprobe. Trypan blue (TB), nitroblue tetrazolium (NBT), acridine orange (AO), fluorescein diacetate (FDA) and ethidium bromide (EB), were used to stain the endothelial layer. SEM was carried out on duplicate samples. Staining with NBT resulted in low cell counts due to loss of cells. There was no significant difference between the extent of damage measured by TB, AO or FDA, but it was shown that staining with FDA and EB can distinguish between damaged and intact cells. Tissue stored for 7 days in a moist chamber had a reduced number of intact cells compared to fresh tissue, and tissue stored by the Capella and Kaufman technique gave a reduced number of intact cells compared to both these control and storage groups. SEM showed surface perforation was characteristic of injured cells, rather than complete disruption. FDA has a theoretical advantage over the other stains, and should provide a more accurate appraisal of defects of cell membrane integrity. For this reason, the use of FDA with EB to stain the endothelium, with SEM carried out on duplicate samples, were preferred as assays to use in the development of corneal storage.

Animals↗